Identification Of Odorant Receptors Responsible For The Perception Of Two Oviposition Attractants In Bactrocera Dorsalis

The oriental fruit fly,Bactrocera dorsalis（Hendel）,is one of the most destructive and invasive pests,causing huge economic losses to the world fruit and vegetable industries.It has been a great challenge to prevent and control this pest due to its strong adaptability,rapid development of insecticide resistance and high fecundity.Currently,the most economic and effective way to control B.dorsalis is olfaction-based trapping.The main component of the widely used commercial attractant is methyl eugenol（ME）which shows strong attraction to the male flies.However,oviposition inside of the fruit is the major reason for fruit damages.Therefore,it would be ideal to develop novel female attractant based control method.Recently,benzothiazole and 1-octen-3-ol have been shown to have the potential to attract females of this fly,especially the gravid females.However,the molecular basis for the perception of benzothiazole and1-octen-3-ol in this fly remains unknown.In this study,we firstly confirmed the involvement of the olfaction system in the perception of benzothiazole and 1-octen-3-ol in B.dorsalis by the EAG and oviposition behavioral assay of the Orco^-/-mutant flies.Subsequently,based on the B.dorsalis antennal transcriptome and genome assembly datasets,we annotated 80 odorant receptor-like（OR-like）genes and detected their expression profiles in different body parts.Meanwhile,we analyzed the induced expression patterns of OR genes after exposure to benzothiazole or 1-octen-3-ol.Meanwhile,we detected the expression profiles of the OR genes in virgin and mated flies.According to the expression profiles,candidate ORs potentially involved in the perception of these two chemicals were screened out for further analysis.Then,voltage clamp recording in Xenopus oocytes as well as the calcium imaging using HEK cell expression system were further performed to detect the binding ability of candidate ORs to benzothiazole and 1-octen-3-ol in vitro.The binding assay data showed that two ORs could bind with benzothiazole and1-octen-3-ol,respectively.Finally,the screened ORs were knocked out by using the CRISPR/Cas9 system.Furthermore,EAG response and oviposition behavior of the mutant flies were detected.Our results provide important theoretical basis for the development of new female oviposition attractants targeting ORs.The main research contents and results are as follows:1.The genome editing of Bdor Orco and phenotypic analysis of the mutantThe structure of Bdor Orco was analyzed based on the genome dataset of B.dorsalis,and the target site of g RNA was selected on the third exon.The synthesized g RNA and purified Cas9 protein were injected into freshly laid eggs.The mosaic G₀was screened based on the polymorphism of PCR products,and the mutant with a 10 bp deletion in Bdor Orco gene was obtained with four generation backcross to the wild type（WT）.Here,the mutant screening strategy was developed to investigate function of genes that do not have obvious morphological changes.EAG responses of Orco^-/-mutant flies to benzothiazole and 1-octen-3-ol were reduced significantly.This indicated that the olfactory sensitivity of mutant flies was significantly decreased.The oviposition assays showed a significant reduction on the egg numbers laid by Orco^-/-mutant flies stimulated by benzothiazole or 1-octen-3-ol.This proved that B.dorsalis recognizes these two oviposition attractants mainly rely on the olfaction system.2.The identification and expression profiling of the ORs in B.dorsalisA total of 20 GB data were obtained from the antennal transcriptome deep sequencing.Based on the amino acid sequences of ORs in Drosophila melanogaster,the t Blastn alignment tool was used to perform homology searching within the B.dorsalis genome,and 80 OR-like genes were identified.The expression of OR-like genes in different body parts including antennae,maxillary palps,head cuticles,probiscis,legs,wings and ovipositors were detected by q PCR.The results showed that OR-like genes were mainly expressed in chemosensory tissues,such as antennae,maxillary palps and head cuticles,while a small amount of OR-like genes were highly expressed in proboscis,such as OR105 and OR119.The odorant induced expression profiles of OR-like genes showed that there were17 and 22 up-regulated genes after benzothiazole and 1-octen-3-ol exposure,respectively.In addition,comparing to the virgin females,there were 24 OR-like genes showed higher expression in mated flies.Taken all the expression profiles together,six OR genes potentially responsible for the perception of benzothiazole（Bdor OR6,Bdor OR18,Bdor OR43a-1,Bdor OR63a-2,Bdor OR13a and Bdor OR5）were screened out,while eight OR genes for 1-octen-3-ol（Bdor OR6,Bdor OR43a-1,Bdor OR13a,Bdor OR5,Bdor OR31,Bdor OR54,Bdor OR60 and Bdor OR108）.3.In vitro binding ability assay of the candidate ORsThe voltage clamp recording in Xenopus oocytes was used to measure the binding ability of candidate ORs to benzothiazole and 1-octen-3-ol in vitro.As the results indicated that Bdor OR43a-1 and Bdor OR63a-2 could be activated by benzothiazole in a dose-dependent manner.The EC₅₀value was 69μM and 12.6μM,respectively.Furthermore,Bdor OR6 and Bdor OR13a also could be activated by 1-octen-3-ol in dose dependent manner with the EC₅₀value of 105μM and 1.27μM,respectively.In addition,calcium imaging using HEK293 cell expression system was further performed to confirm the binding ability of candidate ORs to benzothiazole and1-octen-3-ol.The results also showed that Bdor OR43a-1 and Bdor OR63a-2 could response to benzothiazole and the EC₅₀value was 16.1μM and 2.73μM,respectively.Bdor OR6 and Bdor OR13a could response to 1-octen-3-ol and the EC₅₀value was 15.6μM and 15.03μM,respectively.Homology modeling and molecular docking was performed to explore the theoretical binding site of four important ORs with benzothiazole or 1-octen-3-ol.Auto Dock vina docking results showed that benzothiazole could be combined with SER56 of OR43a-1 as well as LEU181 and TYR287 of OR63a-2,while 1-octen-3-ol could be combined with three amino acids of OR6,ARG16,PHE23 and MET104,and can also be combined with VAL306 and TYR327 of OR13a.4.Mutant screening and phenotypic analysisThe CRISPR/Cas9 system was employed to knock out the four important OR genes,and the homozygous mutants were obtained through four generation backcross to the WT flies.The results showed that OR43a-1^-/-and OR63a-2^-/-mutants showed reduced EAG response to benzothiazole,while OR6^-/-and OR13a^-/-mutants showed reduced EAG response to 1-octen-3-ol.It indicated that the olfaction sensitivity to benzothiazole or 1-octen-3-ol has been significantly reduced in the mutants.The further oviposition of the OR43a-1^-/-and OR63a-2^-/-mutants significantly reduced under benzothiazole exposure,while the oviposition of the OR6^-/-and OR13a^-/-mutants significantly reduced under1-octen-3-ol exposure.In summary,we completed a genome wide OR-like gene annotation for B.dorsalis.We identified the important ORs involved in the perception of benzothiazole and1-octen-3-ol in B.dorsalis,which paved a way to understand the molecular regulation of the odorant mediated oviposition behavior in B.dorsalis.Our results would provide basic information of ORs in B.dorsalis,which will lay a solid foundation for their future function elucidation.In addition,the ORs responsible for benzothiazole and1-octen-3-ol may serve as the target for novel female oviposition attractant development.